MD-2 opsonization enhances phagocytosis and intracellular killing. (A) Human PBMCs were incubated for 30 minutes with Yp and Nm that were left untreated or were opsonized with MD-2^6xHis. Extracellular bacteria were killed by gentamicin treatment, and cells were chased for 6 additional hours in DMEM. Viability of internalized bacteria was assessed by colony count (CFU) and plotted as the average of duplicate determinations plus the range. Similar results were obtained with PBMCs from 2 other unrelated donors. (B) Adherent PBMCs were infected with either untreated or MD-2^6xHis–opsonized YFP-Yp in the absence (SFM) or presence of 30% PHS. Samples were processed and plotted as in Figure 4A. Note the scale difference between the 2 panels. (C) pMφ from the MD-2^−/− mouse were treated as in panel A. (D) pMφ from the MD-2^−/− mouse were infected with untreated or MD-2^6xHis–opsonized Yp in the presence (□) or absence (■) of PHS. Colony count and plotting of live phagocytosed bacteria was performed as in Figure 7A. These experiments were repeated at least 3 times with similar results. Data are averages of triplicates plus SD.